The long term objective of this proposal is to develop a methodology for studying crevicular polymorphonuclear neutrophils (PMN) in health and disease. Specifically, the feasibility of using the fluorescence activated cell sorter (FACS) to study (1) normal peripheral blood PMN and (2) the crevicular PMN function from the same healthy patient will be tested. PMN will be isolated using density gradient centrifugation before FACS analysis. As the analysis progresses, cell populations, will be sorted by the FACS and later stained of microscopic identification. Isolated PMN will be incubated with known stimulators of the respiratory burst such as phorbol esters and with formalin fixed bacteria or with concentrated supernatants from bacterial cultures. After incubation, PMN will be incubated with and without nitroblue tetrazolium (NBT). This study will permit the detection and enumeration of stimulated PMN, which reduce NBT, and thus exhibit light scatter characteristics different from non stimulated PMN. As a final assay of the respiratory burst, stimulated and non stimulated cells will be exposed to laser light at 350 nm, a wavelength at which reduced pyridine nucleotides, found abundantly in stimulated cells, demonstrate a decrease of the naturally occurring fluorescent intensity observed in resting cells. In a second study, PMN will be exposed to chemotactant, FMLP, by attachment of the FMLP to a 0.2 micron fluorescent sphere. Also, neutrophils will be exposed to fluorescent beads that have not been coated with chemotactant. This two part study of the response of normal PMN to chemotactants and the subsequent phagocytosis of particles will provide considerable background information for the study of crevicular PMN. As a control, the reduction of NBT and phagocytosis of fluorescent beads by PMN will also be quantitated and qualitated using microscopic techniques. The analysis of neutrophil response to chemotactants using FACS will be paralleled by more conventional migration chamber studies. The successful completion of this project will result in a technique applicable to the study of neutrophil dysfunction in local and systemic disease. In addition, this methodology will provide a rapid and accurate means of studying the host-plaque interactions found in periodontal diseases.